Immunophenotype of Gastric Tumors Unveils a Pleiotropic Role of Regulatory T Cells in Tumor Development

Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context.

Download Full-text

NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment

Cancers ◽

10.3390/cancers13122999 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2999

Author(s):

Deborah Reynaud ◽

Roland Abi Nahed ◽

Nicolas Lemaitre ◽

Pierre-Adrien Bolze ◽

Wael Traboulsi ◽

...

Keyword(s):

Immune Response ◽

Tumor Cells ◽

Major Gene ◽

Critical Role ◽

Orthotopic Model ◽

Independent Manner ◽

Maternal Immune Response ◽

The Impact

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

Download Full-text

Abstract 152: Stat4 Deficiency limits the Development and Progression of Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a152 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Paresa Taghavie-Moghadam ◽

Matthew Butcher ◽

Mark Kaplan ◽

Jerry Nadler ◽

Elena Galkina

Keyword(s):

T Cells ◽

B Cell ◽

Plaque Burden ◽

Chow Diet ◽

Th1 Cells ◽

Peripheral Tissues ◽

The Impact ◽

Deficient Mice

T helper 1 (Th1) cells constitute the majority of plaque infiltrating IFNγ+ T cells and play a pro-atherogenic role. Th1 cells are induced via IFNγ-dependent activation of T-box expressed in T cells (Tbet) and/or IL-12-dependent activation of signal transducer and activator of transcription 4 (Stat4). While the role of Tbet in atherosclerosis is established, the impact of the IL-12/Stat4-dependent pathway is not well defined. To address the role of Stat4 in atherosclerosis, we bred Stat4-deficient mice with Apolipoprotein E-deficient mice to generate Stat4-/-Apoe-/- mice. Deficiency of Stat4 resulted in approximately a 70% reduction in the plaque burden for 34 week old Stat4-/-Apoe-/- mice fed a chow diet and in 12 week old Stat4-/-Apoe-/- mice fed a western diet there was approximately a 40% reduction in plaque burden, both compared with diet matched Apoe-/- controls females (p<0.001). To assess the effect of Stat4 on Th1 and Treg cell differentiation, we performed an in vitro polarization assay. Deficiency of Stat4 reduced differentiation of IFNγ+ Th1 cells in Th1 conditions, but supported the induction of Tregs in Treg polarizing conditions, confirming the importance of Stat4 in regulating the Th1/Treg balance. In contrast to the in vitro results, we found no difference in the expression of both IFNγ and Foxp3 amongst Stat4-/-Apoe-/- and Apoe-/- lymph nodes and splenic CD4+ T cells; suggesting that additional cytokines in vivo may induce IFNγ+Th1 and inhibit Treg differentiation. Stat4 deficiency also resulted in increased splenic B cell numbers and a slight increase in B1a dependent T15/E06 mRNA expression. Stat4 is a powerful regulator of chemokine expression within peripheral tissues. Adoptively transferred Apoe-/- B cells and CD11b+ cells migrated more efficiently into Stat4-/-Apoe-/- aortas compared to Apoe-/- recipients. However, percentages of macrophages, as determined by CD11b+CD68+ were reduced within the spleens and aortas of Stat4-/-Apoe-/- mice as compared to Apoe-/- controls at steady state conditions. In conclusion, Stat4 deficiency results in reduced atherosclerosis via the modulation of B cell function and aortic leukocyte content.

Download Full-text

Generation and characterization of antitumor infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.145 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 145-145

Author(s):

Juhua Zhou ◽

Yin Zhong ◽

Zhongjun Hou ◽

Jianzhong Zhang ◽

Yanmin Li ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Tumor Cells ◽

Tumor Infiltrating Lymphocytes ◽

Adoptive Cell Transfer ◽

Autologous Tumor ◽

Cell Transfer ◽

Adoptive Cell Transfer Therapy ◽

Infiltrating Lymphocytes

145 Background: Clinical trials have shown that adoptive cell transfer therapy is a promising method for cancer treatment. In the current study, we aim to generate and characterize anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy. Methods: In vitro culture method was used to generate anti-tumor, tumor-infiltrating lymphocytes from patients with breast cancer. FACS analysis, ELISA, and Elispot assay were used to characterize tumor-infiltrating lymphocytes. Autologous anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer were used in adoptive cell transfer therapy. Results: FACS analysis indicated that tumor-infiltrating lymphocytes were present in the tumor tissues, but not detectable in the normal breast tissues from patients with breast cancer. Tumor-infiltrating lymphocytes could be generated in vitro from fresh tumor specimens of patients with breast cancer. Both CD4 T cells and CD8 T cells were detected in tumor-infiltrating lymphocytes. Autologous tumor cells could also generate in vitro from fresh tumor tissue samples of patients with breast cancer. Among 22 samples screened, 6 samples (25%) of tumor-infiltrating lymphocytes are tumor-reactive. Anti-tumor, tumor-infiltrating lymphocytes could recognize autologous tumor cells and allogenic tumor cells. After a large scale T cell expansion, anti-tumor reactivity was maintained in tumor-infiltrating lymphocytes. All of tumor-infiltrating lymphocytes were NK cells in some samples from patients with breast cancer, and these NK cells could recognize autologous tumor cells and a panel of allogenic tumor cells. T cell cloning assay demonstrated that some of the tumor-reactive, tumor-infiltrating lymphocytes were CD4 T cells. Conclusions: The results suggest that anti-tumor, tumor-infiltrating lymphocytes may be generated from patients with breast cancer, which may be used in clinical applications of adoptive cell transfer therapy for patients with breast cancer. The clinical trial of adoptive cell transfer therapy using autologous anti-tumor tumor-infiltrating lymphocytes for patients with breast is under way.

Download Full-text

Addition of immunotherapy in adjuvant setting could be a promising avenue of colorectal cancer (CRC) treatment, especially in patients with higher levels of programmed cell death ligand-1 (PD-L1) positive circulating epithelial tumor cells (CETC) after neoadjuvant chemotherapy.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14036 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14036-e14036

Author(s):

Monika Pizon ◽

Dorothea Schott ◽

Ulrich A. Pachmann ◽

Katharina Pachmann ◽

Siddarth Agrawal ◽

...

Keyword(s):

Neoadjuvant Chemotherapy ◽

Tumor Cells ◽

Tumor Infiltrating Lymphocytes ◽

Histological Subtypes ◽

Epithelial Tumor ◽

Clinicopathologic Factors ◽

Infiltrating Lymphocytes ◽

The Impact ◽

Promising Avenue

e14036 Background: CRC is the third most frequent cancer in men and second in women worldwide. Recently, immune checkpoint molecules such as PD-1/PD-L1 have been identified as a possible target for immunotherapy in CRC and are increasingly being tested in clinical trials in combination with chemotherapy. However, the role of PD-L1 expression in CRC tumor cells and its interaction with other clinicopathologic factors remains elusive. In this study, we evaluated the impact of neoadjuvant chemotherapy on the fraction of PD-L1 positive CETCs and to compare its expression to the primary tumor. Methods: CETCs were determined from the blood of 20 patients suffering from CRC in different stages of the disease. The number of CETCs and their expression of PD-L1 were investigated using the maintrac method. In parallel, PD-L1 expression and tumor infiltrating lymphocytes (TILs) were assessed by immunohistochemistry on tissue biopsies. Results: PD-L1 expression could be assessed in all patients with detectable CETCs with a median of 50%. CETCs were more frequently found to be PD-L1 positive than tissue, and no correlation was observed between tissue and CETCs PD-L1 expression. Interestingly, patients with neoadjuvant chemotherapy had more positive PD-L1 CETCs compared to patients without chemotherapy (mean 66,7 vs. 35,2 p < 0.05), but the total number of CETCs was significantly lower in the neoadjuvant group. Also, patients with mucinous adenocarcinoma had more PD-L1 positive CETCs compared to other histological subtypes. The number of TILs was higher in patients with lymph node metastasis and larger tumors. TILs count did not differ in patients with and without neoadjuvant chemotherapy. Conclusions: Assessment of PD-L1 expression in CETCs is feasible, and CETCs are more often positive than in tissue. PD-L1 expression on CETCs was higher in patients who received neoadjuvant chemotherapy. Further studies are necessary to validate these findings.

Download Full-text

The In Vivo Impact of Human Regulatory T Cells on the Graft Versus Tumor Effect.

Blood ◽

10.1182/blood.v110.11.349.349 ◽

2007 ◽

Vol 110 (11) ◽

pp. 349-349 ◽

Cited By ~ 1

Author(s):

Tuna Mutis ◽

Henk Rozemuller ◽

Maarten E. Emmelot ◽

Tineke Aarts-Riemens ◽

Vivienne Verweij ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Tumor Cells ◽

Cell Reactivity ◽

Human Pbmc ◽

Ifn Γ ◽

T Cell Reactivity ◽

The Impact

Abstract The curative Graft-vs-Tumor effect (GvT) of allogeneic Stem cell transplantation (SCT) is frequently complicated with life threatening Graft-vs-Host Disease (GvHD). In mice, prevention of GvHD, without abrogation of GvT is possible by co-transplantation of naturally occurring regulatory T cells (Tregs) with SC grafts. Consistent with these murine studies, we recently demonstrated that also human Tregs possess potent GvHD-downregulatory capacities in a xenogeneic(x) model, where x-GvHD is induced by infusion of autologous human T cells in RAG2−/−γc−/− mice (Mutis et al. Clin. Cancer Res.2006, 12: 5520–5525). Towards clinical application of Tregs, we now explored the impact of human Treg-administration on GvT in a bioluminescence imaging (BLI) based human-GvT model in the RAG2−/−γc−/− mice. In this model, mice inoculated with luciferase (LUC)-transduced human myeloma (MM) cell lines developed BLI-detectable, progressive, MM-like multifocal tumors exclusively in the bone marrow (BM). Full blown tumors were effectively eliminated by infusion of allogeneic human PBMC. This treatment also caused lethal x-GvHD as expected. In this setting, co-infusion of human PBMC with autologous, in vitro cultured Tregs at a 1:1 Treg: T effector cell ratio had no adverse effects on the development of GvT while significantly reducing the lethality of x-GvHD. In vitro analyses of sacrificed mice at day 21 revealed that administered Tregs homed to BM and spleen, significantly downregulated the total numbers of IFN-γ-producing CD4+ and CD8+ T cells responding to CD3 mediated signals, but had no downregulatory effect on the frequencies of IFN-γ-producing T cells responding to tumor cells. There was also no downregulation of cytotoxic activity against tumor cells in Treg-treated mice. Conclusively, these results showed that Tregs, at doses which are inhibitory for x-GvHD-inducing T cells, could maintain the GvT effect by allowing T cell reactivity against tumor cells. Human Tregs thus still hold promise as attractive cellular tools for separating GvT from GvHD.

Download Full-text

PD-1 suppresses the maintenance of cell couples between cytotoxic T cells and target tumor cells within the tumor

Science Signaling ◽

10.1126/scisignal.aau4518 ◽

2020 ◽

Vol 13 (649) ◽

pp. eaau4518

Author(s):

Rachel Ambler ◽

Grace L. Edmunds ◽

Sin Lih Tan ◽

Silvia Cirillo ◽

Jane I. Pernes ◽

...

Keyword(s):

T Cells ◽

Tumor Cell ◽

Tumor Cells ◽

Ex Vivo ◽

Cytotoxic T Cells ◽

Critical Role ◽

Tumor Infiltrating Lymphocytes ◽

Cell Contact ◽

Similar Phenotype

The killing of tumor cells by CD8+ T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programmed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To find cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8+ tumor-infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL–tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca2+ signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8+ T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell–tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.

Download Full-text

108 MCY-M11, a CAR-PBMC cell product transiently expressing a mesothelin targeted mRNA CAR, exhibits desirable functional and immune phenotype attributed to sustained antitumor immunity in vitro

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0108 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A120-A120

Author(s):

Sashi Kasimsetty ◽

Himavanth Gatla ◽

Dhana Chinnasamy

Keyword(s):

T Cells ◽

Tumor Cells ◽

Antitumor Immunity ◽

Memory T Cells ◽

Cell Populations ◽

Epitope Spreading ◽

Antitumor Response ◽

Cell Product

BackgroundMCY-M11, an anti-mesothelin CAR (Meso-CAR) mRNA transfected PBMC cell product manufactured through <1 day-process is under clinical evaluation for the treatment of advanced ovarian cancer and peritoneal mesothelioma. In this in-vitro study, we characterized the phenotypic and functional status of immune cell populations in MCY-M11 and their possible role in antitumor immunity.MethodsMCY-M11 cell product were generated using unmanipulated healthy donor PBMCs (n=5) by transfection of Meso-CAR mRNA using MaxCyte’s proprietary Flow Electroporation® system. Frozen MCY-M11 cell product was thawed and cultured for 18 hours, then co-cultured with hMSLNneg or hMSLNpos human mesothelioma cell line, MSTO-211H, or stimulated with anti-CD3/anti-CD28 antibodies in vitro for 8 days. Distinct cell populations in MCY-M11 were evaluated for kinetics and duration of CAR expression, differentiation, activation, exhaustion, and their ability to secrete various immunomodulatory molecules during in vitro stimulation. Antigen-specific proliferation and cytotoxicity of MCY-M11 against hMSLNpos tumor cells as well as their ability to mount long-term antitumor immunity through epitope spreading mechanisms were studied.ResultsIndividual cell populations in MCY-M11 exhibited a consistent but transient Meso-CAR expression persisting for about 7 days. Cell subsets in MCY-M11 acquired early signs of activation and differentiation within 18–24 hours post-culture, but only attained full activation and lineage-specific differentiation upon specific response to hMSLNpos tumor cells. hMSLN antigen experienced MCY-M11 retained significant fractions of Naïve and Central Memory T cells and increased percentage of Effector Memory T cells along with increased expression of CD62L, CD27, and chemokine receptors (CCR5, CCR7, and CXCR3). MCY-M11 exhibited strong antigen-specific cytotoxicity against hMSLNpos tumor cells with corresponding increase in activation and proliferation of CD4+ and CD8+ T cell subsets and displayed low or no acquisition of known exhaustion markers. NK cells also exhibited a functionally superior molecular signature exhibiting increased levels of NKG2D, NKp44, NKp46, FAS, and TRAIL. The Monocytes and B cells in MCY-M11 also acquired an activated, differentiated, and mature phenotype, expressing molecules required for antigen presentation (HLA-DR, HLA-ABC, and CD205) and T cell co-stimulation (CD80 and CD86) to mount a strong antitumor response. These phenotypic changes in cell subsets of MCY-M11 transpired with simultaneous secretion of potent immunostimulatory molecules and chemokines facilitating an extended antitumor response through epitope spreading.ConclusionsWe demonstrated that MCY-M11 is a unique cell product possessing a complete built-in immune cellular machinery with favorable phenotype and enhanced functions specialized in mediating an effective and long-term antitumor response.Trial RegistrationNCT03608618

Download Full-text

12 Development of an in vitro assay to assess bispecific T cell engager using T cells from CD3e humanized mice

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0012 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A12-A12

Author(s):

Jun Zhou ◽

Shuang Zhu ◽

Hongjuan Zhang ◽

Lei Zheng ◽

Mingfa Zang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Lymphoblastic Leukemia ◽

Humanized Mice ◽

Activation Markers ◽

In Vivo Models ◽

Vitro Assay ◽

Bite Antibody

BackgroundBispecific T cell engagers (BiTE) is a fast-growing class of immunotherapies. They are bispecific antibody that bind to T cell-surface protein (for example, CD3e) and a specific tumor associate antigen (TAA) on tumor cells, by which to redirect T cells against tumor cells in a MHC-independent manner. A successful example in the clinical is Blinatumomab, a BiTE antibody against CD3/CD19 approved in 2014 to treat acute lymphoblastic leukemia. Currently, many CD3-based BiTE are in clinical trials, including BCMAxCD3, Her2xCD3, CEAxCD3, and PSMAxCD3. To evaluate the efficacy of BiTE in vitro, human peripheral blood monocyte cells (hPBMC) are commonly being used as a source of T cells to co-culture with tumor cells. The disadvantage of using hPBMC is donor-to-donor variability and the availability of the original donor if a study needs to be repeated.MethodsTo overcome this, we proposed to replace hPBMC with T cells from human CD3e (hCD3) genetically engineered mouse models mice (GEMM) for in in vitro coculture assay. T cells were isolated from hCD3 GEMM mice using negative selection mouse T cell isolation kit. Conventional tumor cell lines or luciferase-engineered patient-derived-xenograft (PDX)-derived organoids (PDXO) expressing specific antigens are co-cultured with hCD3 T cells in 96-well plates in the presence of BiTE antibody.ResultsWe measured the killing of tumor cells using either flow cytometry or luciferase activity as readouts. To analyze tumor-reactivity of T cells to cancer cell line or organoids, IFN-gamma in the culture medium was measured and activation markers on T cells was assessed.ConclusionsOur data showed the feasibility of using humanized mice T cells as a replacement for hPBMCs to assess BiTE antibody in vitro. We are further validating the application of murine hCD3 T cells for in vivo models to test bispecific T cell engagers.

Download Full-text

The role of FoxP3+ regulatory T cells and IDO+ immune and tumor cells in malignant melanoma – an immunohistochemical study

BMC Cancer ◽

10.1186/s12885-021-08385-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Satu Salmi ◽

Anton Lin ◽

Benjamin Hirschovits-Gerz ◽

Mari Valkonen ◽

Niina Aaltonen ◽

...

Keyword(s):

T Cells ◽

Prognostic Factors ◽

Regulatory T Cells ◽

Tumor Cells ◽

Immune Cells ◽

Positive Association ◽

Tumor Stage ◽

Melanoma Cells ◽

Melanoma Progression

Abstract Background FoxP3+ Regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) participate in the formation of an immunosuppressive tumor microenvironment (TME) in malignant cutaneous melanoma (CM). Recent studies have reported that IDO expression correlates with poor prognosis and greater Breslow’s depth, but results concerning the role of FoxP3+ Tregs in CM have been controversial. Furthermore, the correlation between IDO and Tregs has not been substantially studied in CM, although IDO is known to be an important regulator of Tregs activity. Methods We investigated the associations of FoxP3+ Tregs, IDO+ tumor cells and IDO+ stromal immune cells with tumor stage, prognostic factors and survival in CM. FoxP3 and IDO were immunohistochemically stained from 29 benign and 29 dysplastic nevi, 18 in situ -melanomas, 48 superficial and 62 deep melanomas and 67 lymph node metastases (LNMs) of CM. The number of FoxP3+ Tregs and IDO+ stromal immune cells, and the coverage and intensity of IDO+ tumor cells were analysed. Results The number of FoxP3+ Tregs and IDO+ stromal immune cells were significantly higher in malignant melanomas compared with benign lesions. The increased expression of IDO in melanoma cells was associated with poor prognostic factors, such as recurrence, nodular growth pattern and increased mitotic count. Furthermore, the expression of IDO in melanoma cells was associated with reduced recurrence˗free survival. We further showed that there was a positive correlation between IDO+ tumor cells and FoxP3+ Tregs. Conclusions These results indicate that IDO is strongly involved in melanoma progression. FoxP3+ Tregs also seems to contribute to the immunosuppressive TME in CM, but their significance in melanoma progression remains unclear. The positive association of FoxP3+ Tregs with IDO+ melanoma cells, but not with IDO+ stromal immune cells, indicates a complex interaction between IDO and Tregs in CM, which demands further studies.

Download Full-text